PROJECT 3: MOLECULAR REGULATION OF HDL METABOLISM AND REVERSE CHOLESTEROL TRANSPORT High density lipoproteins (HDL) and their major protein apolipoprotein A-l (apoA-l) are thought to protect against atherosclerotic cardiovascular disease at least in part by promoting reverse cholesterol transport (RCT), whereby excess cholesterol is removed from the periphery (such as the vascular wall) and returned to the liver for excretion. The broad goal of this project is to generate greater understanding of the molecular physiology of HDL metabolism as it relates to reverse cholesterol transport (RCT) by using integrated in vivo models in mice and extending experiments where possible into humans. It has become clear that the plasma concentration of HDL cholesterol (HDL-C) is not an adequate surrogate for anti-atherogenic effects, and that measures of cholesterol flux through the RCT pathway and of HDL function are more critical with regard to effects on cardiovascular disease. Specific Aim 1 will involve the use of mouse in vivo experiments to study the structure-function properties of apoA-l, in studies that complement the physico- chemical studies in Project 2 and the cell-based studies in Project 1. ApoA-l variants will be inserted into AAV8-based gene expression vectors, which will be used to express these variant apoA-l molecules in apoA-l knockout mice and effects on HDL metabolism, RCT, and in selected cases, atherosclerosis will be determined. Specific Aim 2 will address, using mouse models, the roles of lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP) in modulating the rate of macrophage RCT and the relationship to atherogenesis. Specific Aim 3 will extend concepts and approaches developed in mice into the human setting, with the specific goal of measuring RCT using new methods in humans with Tangier disease (ABCA1 deficiency) and with LCAT deficiency. While high levels of HDL cholesterol are associated with reduced risk of coronary artery disease, it is not clear that simply raising levels of HDL cholesterol are sufficient to reduce cardiovascular risk. The mechanisms by which HDL is altered and the impact on the process of reverse cholesterol transport is likely to be a more important determinant of cardiovascular risk. This project will seek to understand the regulation of reverse cholesterol transport in living mice and humans. University of Pennsylvania School of Medicine Institute for Translational Medicine and Therapeutics 654 BRBII/III 421 Curie Blvd Philadelphia, PA 19104 PHS 398 (Rev. 04/06) Form Page 2 173 Principal Investigator/Program Director (Last, First, Middle): Phillips, Michael C. PROJECT 3 KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator(s). List all other key personnel in alphabetical order, last name first. Name Rader, Daniel J. Professor of Medicine, Pathology and Pharmacology Cuchel, Marina Research Assistant Prof, of Medicine OTHER SIGNIFICANT CONTRIBUTORS Name Chaitanya Divgi Alan Remaley eRA Commons User Name Organization Role on Project Universityof DANRADER PennsylvaniaSchool of PI Medicine University of BILLHEIJ Universityof MCUCHEL Pennsylvania School of Co-Investigator Medicine Organization Role on Project University of Pennsylvania Collaborator NIH/NHLBI Collaborator Human Embryonic Stem Cells X No Q Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://Stemcells.nih.qov/reqistry/index.asp. Use continuation pages as needed. If a specificline cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Form Page 2-continued 174 Principal Investigator / Program Director (Last. First. Middle): Phillips. Michael C. PROJECT 3 FROM THROUGH DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY 12/01/08 11/30/09 PERSONNEL (Applicant organization only) Months Devoted to Proiect ROLE ON Cal. Acad. Summer NAME PROJECT Mnths Mnths Mnths Principal Rader, Daniel Investigator 2.40 Billheimer, Jeffrey Co-Investigator 1.20 Cuchel, Marina Co-Investigator 0.6 Postdoctoral Tanigawa, Hiroyuki 12.0 Fellow Research Deborah Cromley 3.0 Specialist Research AishaWilson 6.0 Specialist Page 2 SUBTOTALS CONSULTANT COSTS EQUIPMENT (Itemize) SUPPLIES (Itemize) Plastic/Glassware Chemicals, radiochemicals and biological reagents Clinical supplies and investigational drugs Animal purchase TRAVEL PATIENT CARE COSTS INPATIENT OUTPATIENT ALTERATIONS AND RENOVATIONS (Itemize by category) OTHER EXPENSES (Itemize by category) Animal Per Diems Post doc health insurance Publications Subject Comoensation CONSORTIUM/CONTRACTUAL COSTS DOLLAR AMOUNT REQUESTED (omit cents) INST.BASE SALARY FRINGE SALARY REQUESTED BENEFITS TOTAL **** 37,320 11,159 48,479 97,600 9,760 2,918 12,678 86,570 0 0 0 55,426 55,426 5,376 60,802 62,588 15,647 4,678 20,325 43,894 21,947 6,562 28,509 26,252 7,850 34,102 ^w 166,352 38,543 204,895 7,013 10,000 4,000 10,000 31,013 10,000 8,100 2,500 12.500 33,100 DIRECT COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a. Face Page) $ 269 008 CONSORTIUM/CONTRACTUAL COSTS FACILITIES ANDADMINISTRATIVE COSTS 154,679 TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 423 687 PHS 398 (Rev. 04/06) Form Page 4 175